The Northwest Metabolomics Research Center (NW-MRC) at the University of Washington in Seattle is the largest comprehensive metabolic profiling laboratory in the Pacific Northwest Region. We have developed a robust and reproducible targeted mass spectrometry platform that measures more than 200 aqueous metabolites and is in very high demand. This platform is based on an AB-Sciex API5500 Qtrap mass spectrometer coupled to Agilent 1260 dual liquid chromatography pumps that separate aqueous metabolites using HILIC columns under two identical conditions for both positive and negative ionization. Due to its excellent measurement reproducibility and robustness, this assay has attracted a very high interest from basic and clinical researches. For example, since the beginning of 2014, we have analyzed over 3,500 biological samples from more than 60 collaborators on this system, and the results have been reported in 10 peer-reviewed articles. At this point, demand for the system is exceeding our capacity, and with the strong growth we have experienced, we anticipate longer and longer delays in running samples. In addition, we would like to add targeted lipidomics to our assay, but do not have instrument time to devote for method development or for running the additional lipids on this platform. Finally, we have developed a new approach for metabolomics, globally optimized targeted (GOT)-MS, which combines the benefits of global and targeted analysis and promises very broad metabolome coverage with high reproducibility. However, our AB-Sciex system is the only one we have that can perform targeted measurements of over 200 molecules with good long term stability. Given the high demand for this system, we greatly need the addition of a more versatile and sensitive LC-MS/MS system for advanced targeted metabolomics. To further enhance targeted metabolomics capabilities we propose to install a state-of-the-art multiplexed UPLC- MS/MS system capable of performing simultaneous separations on four analytical columns followed by high sensitive targeted MS acquisition. Each column will be independently connected to a 4-port injection auto- sampler, which in turn will allow separations under four different chromatography conditions. The major benefits of this platform will be: (i) much broader coverage of measured metabolites since we will add hundreds of lipids to our targeted assay; (ii) significant enhancement in the throughput capabilities, which in turn will eliminate delays in analysis turnaround time; (iii) additional time available for method development. As a result, we will be able to develop and utilize our new GOT-MS methodology to improve metabolite measurements. The proposed system will be available for collaborative research projects at least 75% of the run time, and its extensive utilization will lead to a better understanding of metabolic processes associated with disease development, biomarker discovery, and systems biology research.